Microtubule-associated phase separation of MIDD1 tunes cell wall spacing in xylem vessels in Arabidopsis thaliana.

Properly patterned cell walls specify cellular functions in plants. Differentiating protoxylem and metaxylem vessel cells exhibit thick secondary cell walls in striped and pitted patterns, respectively. Cortical microtubules are arranged in distinct patterns to direct cell wall deposition. The scaffold protein MIDD1 promotes microtubule depletion by interacting with ROP GTPases and KINESIN-13A in metaxylem vessels. Here we show that the phase separation of MIDD1 fine-tunes cell wall spacing in protoxylem vessels in Arabidopsis thaliana. Compared with wild-type, midd1 mutants exhibited narrower gaps and smaller pits in the secondary cell walls of protoxylem and metaxylem vessel cells, respectively. Live imaging of ectopically induced protoxylem vessels revealed that MIDD1 forms condensations along the depolymerizing microtubules, which in turn caused massive catastrophe of microtubules. The MIDD1 condensates exhibited rapid turnover and were susceptible to 1,6-hexanediol. Loss of ROP abolished the condensation of MIDD1 and resulted in narrow cell wall gaps in protoxylem vessels. These results suggest that the microtubule-associated phase separation of MIDD1 facilitates microtubule arrangement to regulate the size of gaps in secondary cell walls. This study reveals a new biological role of phase separation in the fine-tuning of cell wall patterning.

Confined-microtubule assembly shapes three-dimensional cell wall structures in xylem vessels.

Properly patterned deposition of cell wall polymers is prerequisite for the morphogenesis of plant cells. A cortical microtubule array guides the two-dimensional pattern of cell wall deposition. Yet, the mechanism underlying the three-dimensional patterning of cell wall deposition is poorly understood. In metaxylem vessels, cell wall arches are formed over numerous pit membranes, forming highly organized three-dimensional cell wall structures. Here, we show that the microtubule-associated proteins, MAP70-5 and MAP70-1, regulate arch development. The map70-1 map70-5 plants formed oblique arches in an abnormal orientation in pits. Microtubules fit the aperture of developing arches in wild-type cells, whereas microtubules in map70-1 map70-5 cells extended over the boundaries of pit arches. MAP70 caused the bending and bundling of microtubules. These results suggest that MAP70 confines microtubules within the pit apertures by altering the physical properties of microtubules, thereby directing the growth of pit arches in the proper orientation. This study provides clues to understanding how plants develop three-dimensional structure of cell walls.

A Quantitative Method for Evaluating Phragmoplast Morphology.

Phragmoplasts are plant-specific microtubule structures that form cell plates at the cell division plane. During late anaphase, phragmoplasts emerge between daughter nuclei as the derivative of spindle microtubules, and centrifugally expand toward the cell cortex to build cell plates during telophase. Phragmoplasts are composed of short antiparallel microtubules decorated with various microtubule-associated proteins. Mutants of these microtubule-associated proteins exhibit defects in phragmoplast morphology. Quantification of phragmoplast morphology is indispensable for assessing the phenotypes of these mutants. Here, we describe a method to quantify the width of phragmoplasts.

A Novel Katanin-Tethering Machinery Accelerates Cytokinesis.

Cytokinesis is fundamental for cell proliferation [1, 2]. In plants, a bipolar short-microtubule array forms the phragmoplast, which mediates vesicle transport to the midzone and guides the formation of cell walls that separate the mother cell into two daughter cells [2]. The phragmoplast centrifugally expands toward the cell cortex to guide cell-plate formation at the cortical division site [3, 4]. Several proteins in the phragmoplast midzone facilitate the anti-parallel bundling of microtubules and vesicle accumulation [5]. However, the mechanisms by which short microtubules are maintained during phragmoplast development, in particular, the behavior of microtubules at the distal zone of phragmoplasts, are poorly understood. Here, we show that a plant-specific protein, CORTICAL MICROTUBULE DISORDERING 4 (CORD4), tethers the conserved microtubule-severing protein katanin to facilitate formation of the short-microtubule array in phragmoplasts. CORD4 was specifically expressed during mitosis and localized to preprophase bands and phragmoplast microtubules. Custom-made two-photon spinning disk confocal microscopy revealed that CORD4 rapidly localized to microtubules in the distal phragmoplast zone during phragmoplast assembly at late anaphase and persisted throughout phragmoplast expansion. Loss of CORD4 caused abnormally long and oblique phragmoplast microtubules and slow expansion of phragmoplasts. The p60 katanin subunit, KTN1, localized to the distal phragmoplast zone in a CORD4-dependent manner. These results suggest that CORD4 tethers KTN1 at phragmoplasts to modulate microtubule length, thereby accelerating phragmoplast growth. This reveals the presence of a distinct machinery to accelerate cytokinesis by regulating the action of katanin.

Imaging of Developing Metaxylem Vessel Elements in Cultured Hypocotyls.

An in vitro induction system for xylem vessel formation is a useful tool for visualizing the differentiation of xylem vessel cells. A procedure for inducing xylem vessel cell differentiation in hypocotyls of Arabidopsis thaliana is described here. Metaxylem vessel elements form ectopically in excised hypocotyl tissue following treatment with bikinin. This enables high-resolution imaging of living metaxylem vessel cells. The wide range of resources available for Arabidopsis allows for the visualization of diverse cellular structures, including microtubules and secondary cell walls, in different genetic backgrounds. Use of this system will contribute to the further understanding of the processes by which xylem vessel elements form.

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels.

Rho GTPases play crucial roles in cell polarity and pattern formation. ROPs, Rho of plant GTPases, are widely involved in cell wall patterning in plants, yet the molecular mechanism underlying their action remains unknown. Arabidopsis ROP11 is locally activated to form plasma membrane domains, which direct formation of cell wall pits in metaxylem vessel cells through interaction with cortical microtubules. Here, we show that the pattern formation of cell wall pits is governed by ROP activation via a reaction-diffusion mechanism. Genetic analysis and reconstructive assays revealed that ROPGEF4/7 and ROPGAP3/4, which encode ROP activators and inactivators, respectively, regulated the formation of ROP-activated domains; these in turn determined the pattern of cell wall pits. Mathematical modelling showed that ROP-activation cycle generated ROP domains by reaction-diffusion mechanism. The model predicted that a positive feedback and slow diffusion of ROP11-ROPGEF4 complex were required to generate ROP-activated domains. ROPGEF4 formed a dimer that interacted with activated ROP11 in vivo, which could provide positive feedback for ROP activation. ROPGEF4 was highly stable on the plasma membrane and inhibited ROP11 diffusion. Our study indicated that ROP-based reaction-diffusion system self-organizes ROP-activated domains, thereby determines the pit pattern of metaxylem vessels.

CORTICAL MICROTUBULE DISORDERING1 Is Required for Secondary Cell Wall Patterning in Xylem Vessels.

Proper patterning of the cell wall is essential for plant cell development. Cortical microtubule arrays direct the deposition patterns of cell walls at the plasma membrane. However, the precise mechanism underlying cortical microtubule organization is not well understood. Here, we show that a microtubule-associated protein, CORD1 (CORTICAL MICROTUBULE DISORDERING1), is required for the pitted secondary cell wall pattern of metaxylem vessels in Arabidopsis thaliana Loss of CORD1 and its paralog, CORD2, led to the formation of irregular secondary cell walls with small pits in metaxylem vessels, while overexpressing CORD1 led to the formation of abnormally enlarged secondary cell wall pits. Ectopic expression of CORD1 disturbed the parallel cortical microtubule array by promoting the detachment of microtubules from the plasma membrane. A reconstructive approach revealed that CORD1-induced disorganization of cortical microtubules impairs the boundaries of plasma membrane domains of active ROP11 GTPase, which govern pit formation. Our data suggest that CORD1 promotes cortical microtubule disorganization to regulate secondary cell wall pit formation. The Arabidopsis genome has six CORD1 paralogs that are expressed in various tissues during plant development, suggesting they are important for regulating cortical microtubules during plant development.

Shoot-derived cytokinins systemically regulate root nodulation.

Legumes establish symbiotic associations with nitrogen-fixing bacteria (rhizobia) in root nodules to obtain nitrogen. Legumes control nodule number through long-distance communication between roots and shoots, maintaining the proper symbiotic balance. Rhizobial infection triggers the production of mobile CLE-RS1/2 peptides in Lotus japonicus roots; the perception of the signal by receptor kinase HAR1 in shoots presumably induces the production of an unidentified shoot-derived inhibitor (SDI) that translocates to roots and blocks further nodule development. Here we show that, CLE-RS1/2-HAR1 signalling activates the production of shoot-derived cytokinins, which have an SDI-like capacity to systemically suppress nodulation. In addition, we show that LjIPT3 is involved in nodulation-related cytokinin production in shoots. The expression of LjIPT3 is activated in an HAR1-dependent manner. We further demonstrate shoot-to-root long-distance transport of cytokinin in L. japonicus seedlings. These findings add essential components to our understanding of how legumes control nodulation to balance nutritional requirements and energy status.